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Blood, Vol. 112, Issue 10, 4048-4050, November 15, 2008
Plasmin therapy enhances mobilization of HPCs after G-CSF
Blood Tjwa et al.
112: 4048
Supplemental materials for: Tjwa et al
EXPERIMENTAL METHODS Animal studies
Mice lacking PAI-1 (75% Bl6 × 25% 129Sv) or AP (50% Bl6 × 50% 129Sv) and their respective WT littermates were used.1 For all experiments, age-, gender- and strain-matched mice were used. Mice were maintained in HEPA-filtered IVC units. All experiments were performed according to the guidelines for care and use of laboratory animals approved by the institutional ethical animal care committee. Mice were injected with G-CSF (200 µg/kg/d, Filgrastrim, Amgen) s.c. for 5 consecutive days. Peripheral blood was repetitively sampled by retro-orbital puncture under light anesthesia, and full blood counts (EDTA buffered) were determined on a hemocytometer (Cell-Dyn 1300, Abbott). Tenecteplase (TNK; 100 mg/kg; Metalyse®, Boehringer Ingelheim), recombinant human tPA (100 mg/kg; Actilyse®) or solvent was administered via daily intra-peritoneal bolus injections. Micro-plasmin (100 µg/day, Thrombogenics; endotoxin levels < 0.5 EU/mg protein) or vehicle was administered via osmotic minipumps (Alzet 2001). HPC assays and transplantation experiments
Mononuclear cells (MNCs) from PB or BM were prepared via Lympholiter-M (Cedarlane, Sanbio) and density centrifugation. For CFU-C assays, BM cells (5 × 104), or PB-MNCs (1 × 105) were plated in 35 mm dishes (Stem Cell Technologies) using methylcellulose supplemented with growth factors (MethoCult, Stem Cell Technologies), and colonies were blindly scored after 7 and 13 days, respectively, using an inverted microscope. For CFU-S assays, lethal total body irradiation (9.0 to 9.5 Gy) and transplantation of mobilized PB-MNCs (1 to 1.5 × 105) were performed in syngeneic WT recipients, survival was monitored or splenic colonies were blindly scored after 12 days using a dissection microscope. Patient study
Peripheral blood samples (EDTA anti-coagulated) were collected after informed consent from patients admitted to the CCU for thrombolytic or PCI treatment because of clinical and biochemical signs of acute coronary syndrome (STEMI: ST elevation on ECG and Troponin-T+). Plasma was collected, and MNCs were prepared via Lymphoprep (Axis-Shield, Lucron, De Pinte, Belgium) and density centrifugation. For CFU-C assays, PB-MNCs (1 × 105 and 5 × 105) were plated in 35 mm dishes (Stem Cell Technologies) using methylcellulose supplemented with growth factors (MethoCult, Stem Cell Technologies), and colonies were blindly scored after 13 days using an inverted microscope. For FACS analysis, 1 × 106 MNCs were stained with anti-human CD34 or isotype antibodies (Tebu Bio), and CD34+ stem/progenitors were quantified using appropriate gating. To prevent non-specific binding, FcR blocking reagent (Tebu Bio) was added. The protocol of this non-randomized clinical study was approved by the local medical ethical committee (ML2526). Statistics
We used SPSS v. 11.0 for statistical calculations. Unless stated otherwise, data (mean ± SEM) were statistically analyzed by an unpaired Student’s t-test. Cox regression was used to analyze the genotypic differences in survival. P<0.05 was considered statistically significant. REFERENCES 1. Dewerchin M, Collen D, Lijnen HR. Enhanced fibrinolytic potential in mice with combined homozygous deficiency of alpha2-antiplasmin and PAI-1. Thromb Haemost. 2001;86:640-646.
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